Sample: CBA.G4F-426-L3W

Analytes:

Overview

The gel image below shows the locations of the analyte chains presented in this report.

Alpha-1-antitrypsin (P01009)

From: Human plasma protein N-glycosylation

Alpha-1-antitrypsin (AAT), also known as alpha-1-protease inhibitor, alpha-1-antiproteinase or serpin A1, consists of 418 amino acids (including a 24 amino acid signal peptide) with an apparent mass of 51 kDa (including glycosylation). It is mainly produced in the liver by hepatocytes, but is also synthesized in monocytes, intestinal epithelial cells, and in the cornea [52, 208–211]. Due to its small size and polar properties, the glycoprotein can easily move into tissue fluids [52]. In healthy individuals, a plasma level of approximately 1.1 mg/mL is found, but the concentration can increase three- to four-fold during inflammation [212–215]. AAT occurs as three different amino acid sequences, of which the first is set as the standard sequence. Form 2 differs in the amino acid sequence 356–418 and form 3 lacks the amino acid sequence 307–418.

Apolipoprotein D (P05090)

From: Human plasma protein N-glycosylation

Apolipoprotein D (Apo D), also referred to as thin line polypeptide, is a small glycoprotein of 189 amino acids (with a signal peptide of 20 amino acids), with a molecular weight varying between 19 and 32 kDa depending on its glycosylation [262, 263]. While it shares their name, it does in fact not resemble other apolipoproteins, and shares more homology with the lipocalin protein family [264]. It was originally assimilated to the apolipoprotein family due to its early association with lipid transport. Apo D is mainly synthetized in fibroblasts and to a lesser extent in the liver and intestine, where the other apolipoproteins are usually produced [265]. Its plasma levels are approximately 0.1 mg/mL [266, 267]. The common form of Apo D in plasma is a monomer, although it can also exist as a heterodimer linked to apolipoprotein A-2 via a disulfide bridge. Apo D can form complexes with lecithin cholesterol acyltransferase and is implicated in the transport and transformation of lipids [264, 268–270]. It has been reported to have a potential role in colorectal cancer [265]. In addition, the protein is present at high concentrations in the cyst fluid where its concentration can be 500 times higher than in plasma, which can be associated with an increased risk of breast cancer [271–273]. Apo D has a tendency to accumulate in CSF and peripheral nerves of patients with Alzheimer’s disease and other neurodegenerative conditions [274, 275]. A positive correlation between age and Apo D levels has been reported in females, but not in men [276, 277].

Clusterin (P10909)

From: UniProt

Functions as extracellular chaperone that prevents aggregation of non native proteins (PubMed:11123922, PubMed:19535339). Prevents stress-induced aggregation of blood plasma proteins (PubMed:11123922, PubMed:12176985, PubMed:17260971, PubMed:19996109). Inhibits formation of amyloid fibrils by APP, APOC2, B2M, CALCA, CSN3, SNCA and aggregation-prone LYZ variants (in vitro) (PubMed:12047389, PubMed:17412999, PubMed:17407782). Does not require ATP (PubMed:11123922). Maintains partially unfolded proteins in a state appropriate for subsequent refolding by other chaperones, such as HSPA8/HSC70 (PubMed:11123922). Does not refold proteins by itself (PubMed:11123922). Binding to cell surface receptors triggers internalization of the chaperone-client complex and subsequent lysosomal or proteasomal degradation (PubMed:21505792). Protects cells against apoptosis and against cytolysis by complement (PubMed:2780565). Intracellular forms interact with ubiquitin and SCF (SKP1-CUL1-F-box protein) E3 ubiquitin-protein ligase complexes and promote the ubiquitination and subsequent proteasomal degradation of target proteins (PubMed:20068069). Promotes proteasomal degradation of COMMD1 and IKBKB (PubMed:20068069). Modulates NF-kappa-B transcriptional activity (PubMed:12882985). A mitochondrial form suppresses BAX-dependent release of cytochrome c into the cytoplasm and inhibit apoptosis (PubMed:16113678, PubMed:17689225). Plays a role in the regulation of cell proliferation (PubMed:19137541). An intracellular form suppresses stress-induced apoptosis by stabilizing mitochondrial membrane integrity through interaction with HSPA5 (PubMed:22689054). Secreted form does not affect caspase or BAX-mediated intrinsic apoptosis and TNF-induced NF-kappa-B-activity (PubMed:24073260). Secreted form act as an important modulator during neuronal differentiation through interaction with STMN3 (By similarity). Plays a role in the clearance of immune complexes that arise during cell injury (By similarity).

   Clusterin / Apolipoprotein J (clusters)

Ceruloplasmin (P00450)

From: Human plasma protein N-glycosylation

Ceruloplasmin (CP), also called ferroxidase, is a 132 kDa (120 kDa without glycosylation) 1065 amino acid (19 of which are signal peptide) glycoprotein synthesized by the liver [103]. It consists of a single polypeptide chain, and belongs to the multicopper oxidase family [103]. Concentrations for CP range from 0.15 to 0.96 mg/mL with a mean of 0.36 mg/mL, while elevated levels have been reported upon inflammatory stimulation [34, 288, 289]. CP can bind six to seven atoms of copper, in this manner containing and transporting 95 % of the copper found in plasma. The main function of the protein, however, is in iron metabolism. CP has ferroxidase activity oxidizing Fe2+ to Fe3+ without releasing radical oxygen species, while also facilitating iron transport across the cell membrane.

Fibrinogen (P02671; P02675; P02679)

From: Human plasma protein N-glycosylation

Fibrinogen is a 340 kDa glycoprotein that is synthesized in the liver by hepatocytes, and plays a key role in blood clotting [290, 291]. The protein consists of two sets of three different polypeptide chains named the a-chain (610 amino acids), ß-chain (461 amino acids), and <U+03B3>-chain (411 amino acids), arranged in a a2ß2<U+03B3>2 hexamer linked by disulfide bonds [106, 292, 293]. In plasma, fibrinogen is typically found at concentrations of 2–6 mg/mL with a mean of 3 mg/mL, with women having slightly higher levels, and it is also present in platelets, lymph nodes, and interstitial fluid [106, 293–296]. Fibrinogen is cleaved by thrombin into fibrin, one of the essential components of blood clots after injury [106, 291, 297]. Furthermore, it acts as a cofactor in platelet aggregation, assists rebuilding of epithelium, and can protect against infections in interferon <U+03B3> (IFN<U+03B3>)-mediated hemorrhage [106, 298, 299]. In addition, the protein can facilitate the immune response via the innate and T-cell pathways [300–303].

Haptoglobin (P00738)

From: Human plasma protein N-glycosylation

Haptoglobin (Hp) is a 406 amino acid (18 amino acid signal peptide) acute-phase glycoprotein with a peptide backbone of 45 kDa. It is synthesized in the liver by hepatocytes as a single polypeptide chain and is also found in skin [304, 305]. During its synthesis, Hp is cleaved into a light a chain and a heavy ß chain that are connected via disulfide bonds. Two variants of the a chain originating from the sequence Val19-Gln160 and differing by the subsequence Glu38-Pro96 can exist, a1 having this subsequence once while a2 has it twice, resulting in a chains of 83 or 142 amino acids with a respective molecular mass of 9 and 16 kDa. The 40 kDa ß chain is made of 245 amino acids originating from the sequence Ile162-Asn406 [306, 307]. The combination of different allelic variants of the a chain (a1 and a2) with ß chain(s) creates the polymorphism observed in Hp. There are three major Hp phenotypes called Hp1-1, Hp2-1 and Hp2-2. They have a configuration of (a1ß)2, (a1ß)2 + (a2ß)n = 0, 1, 2, … and (a2ß)n = 3, 4, 5, …, respectively, which are observed at different ratios among ethnicities [118, 308–310]. Caucasians have around 13 % of phenotype Hp1-1, 46 % of Hp2-1 and 41 % of Hp2-2. Hp is typically found at a plasma levels in the range of 0.6–2.3 mg/mL with a mean of 1.32 mg/mL [118]. Elevated Hp levels have been reported with inflammation and malignant diseases [308, 311, 312]. It should be taken into account that the concentration as well as the molecular mass including glycosylation may vary among phenotypes (86–900 kDa) [118]. The half-life of Hp is found to be on average four days.

The major function of Hp is to protect tissues from oxidative damage by capturing hemoglobin [307, 313]. It has been reported that Hp polymorphism has an effect on its physiological properties, for instance Hp1-1 binds hemoglobin stronger than Hp2-2 [314]. Certain diseases seem to be dependent on the polymorphism, as individuals with the Hp1-1 phenotype seem to have a higher concentration of induced antibodies in their plasma after vaccination, infections or liver diseases compared to the other phenotypes [118, 310].

   Haptoglobin Alpha Chain (clusters)

Immunoglobulin G (P01857; P01859; P01860; P01861)

From: Human plasma protein N-glycosylation

Immunoglobulin gamma (IgG) is a glycoprotein with a total molecular mass of approximately 150 kDa [159, 177]. The light chain consists of a domain covering the variable region (VL) as well as a constant (CL) domain. The heavy chain contains four domains with one domain which comprises the variable region (HL) followed by three constant domains: CH1, CH2 and CH3. Each light chain is paired with the HL and CH1 domain of the heavy chain to form a Fab portion, whilst CH2 and CH3 domains of the two heavy chains together form the Fc portion. Between the two Fab portions and the Fc portion a flexible hinge region is positioned, which makes it possible for the two Fab arms to move individually [159, 364]. The antibody is highly stable with a half-life of approximately 12 days [369, 370]. Based on the amino acid sequence of the constant regions of the heavy chains, IgGs can be divided into four subclasses namely, IgG1 (P01857), IgG2 (P01859), IgG3 (P01860) and IgG4 (P01861) [371, 372]. Notably, IgG3 has a larger hinge region (62 amino acids) compared to the other subclasses (12 amino acids). During a secondary immune response IgG is secreted in high amounts by B cells [373]. In healthy individuals the concentration of IgG in serum is between 7 and 18 mg/mL [374]. The average subclass-specific concentrations in plasma are reported as 5.03 mg/mL for IgG1, 3.42 mg/mL for IgG2, 0.58 mg/mL for IgG3 and 0.38 mg/mL for IgG4 [375]. IgG molecules are important for activating the complement system through the classical pathway (antibody-triggered) as well as binding to specific receptors on macrophages and neutrophils [364, 373]. The IgG subclasses differ in their ability to activate the complement system. The primary activators of the complement system are IgG1 and IgG3, whereas IgG2 can also activate it at a lower level. IgG4, on the other hand, is not capable of activating the complement system [364]. Furthermore, IgG molecules are the only antibodies that can pass from a mother to her child via the placenta, and maternal IgG has been shown to gradually decrease throughout pregnancy [364, 376, 377]. The majority of therapeutic antibodies is derived from IgG1, where the glycosylation plays an important role for their function [35, 364, 377–380].

Plasminogen (P00747)

Transferrin (P02787)

From: Human plasma protein N-glycosylation

Serotransferrin (STF), also known as transferrin, ß1 metal binding globulin or siderophilin, is a 698 amino acid protein (19 amino acids of which are signal peptide) with a molecular mass of approximately 77 kDa (without glycosylation) [8, 337]. The protein consists of two globular domains, the N-lobe and the C-lobe which divided into two subdomains each (N1, N2, C1 and C2). The two main domains are connected by a short linker peptide [337–339]. The N-lobe is 336 amino acids in size and spans from Val25 to Glu347, while the C-lobe is 343 amino acids long and ranges from Val361 to Lys683 [337]. The lobes can interact to form a hydrophilic metal ion binding site [337]. STF is mostly produced by hepatocytes, although other tissues have also shown expression, albeit at significantly lower amounts [337]. The plasma concentration is highly stable from the age of 2 years on, with a range between 2 and 3 mg/mL [337, 340]. Levels may increase during pregnancy up to 5 mg/mL [141].

Vitamin D-binding protein (VTDB) (P02774)

From: UniProt

Involved in vitamin D transport and storage, scavenging of extracellular G-actin, enhancement of the chemotactic activity of C5 alpha for neutrophils in inflammation and macrophage activation.

   Vitamin D-Binding Protein (clusters)

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Statistical Process Control

From: An introduction to statistical process control in research proteomics

Background: Statistical process control is a well-established and respected method which provides a general purpose, and consistent framework for monitoring and improving the quality of a process. It is routinely used in many industries where the quality of final products is critical and is often required in clinical diagnostic laboratories [1,2]. To date, the methodology has been little utilised in research proteomics. It has been shown to be capable of delivering quantitative QC procedures for qualitative clinical assays [3] making it an ideal methodology to apply to this area of biological research.

Control charts and possibly image areas from the standard.

Audit Logs:

 Run overview

Run #	208
Sample / Study	Human plasma (Lot: SLCC1673)/ NUBZ64100, NUCX182416, NUBZ648698, NUAQ387360, NUFH402412, NUFT865086, NUFR670624, NUFT766262, NUFU715690, NUFT770122, NUFT766280, NUCX210223
Completion (Scan) Date	25/09/2020
Comments	OOD GE strips, biotium dye, sample load 2uL. Pre-mix buffers. Standard IEF with cuploading. Standard 2D with type 2 water. 4% equilibration buffer.

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 Labelling

Technician:EA & NH

Sample: 2µL Human Plasma

Solution / reagent	Batch / Lot #	Reference
DIGE labelling buffer	6	LB-RS-06
Tris-HCl pH 8.5	2	LB-RS-02
DMF	STBH7014
Cy3 (Biotium)	13C1212
Cy5 (Biotium)	18C0626-1075
Lysine	4	LB-RS-07
2x DIGE buffer	6	LB-RS-04

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 Iso Electric Focusing

Technician:EA & NH

Solution / reagent	Batch / Lot #
1x DIGE buffer	14
Control sample	Human plasma/ SLCC1673
IPG strips	10277023
Reswell Date	22/09/2020
Time strip 1	16:10:00
Time oil added	16:40:00

Equipment	ID #
Reswell tray	3
IPGphor unit	1

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 2D SDS

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 Imaging

Technician: EA & NH

Imager: Typhoon 9400

Calibration App Log File

The imager was calibrated on 25/09/2020 following protocol P-212 and Calibration App Version 1.01.

Dye channel	PMT
Cy5	450
Cy3	455

Scanner Driver App

The gels were imaged in pairs using Scanner Driver App version 1.01. The app automatically cropped and rotated the scans. Scans were performed at 100 and 200 micron resolutions. Cy3 and Cy5 channels were imaged simultaneously.

Whole gel visual review

The gels passed a visual review (under OP-28) before upload to the cloud for processing.

Scanning App Log File

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